Imaging multiple chemical reactions in a flow cell array

ABSTRACT

Systems, computer program products, and methods for using a flow cell array are provided herein. A computer program product includes a computer readable storage medium having program instructions embodied therewith, the program instructions executable by a device to cause the device to determine placement of multiple reaction site openings, wherein each reaction site opening is connected to a first sub-surface channel; connect the first sub-surface channel to two or more additional sub-surface channels by multiple vias; and provide a material for multiple reaction sites, wherein an overlap of the multiple reaction site openings and the material delineate the multiple reaction sites.

FIELD OF THE INVENTION

Embodiments of the invention generally relate to information technology,and, more particularly, to biological sequencing.

BACKGROUND

Existing deoxyribonucleic acid (DNA) sequencing techniques, such assequencing-by-synthesis (SBS), sequencing-by-ligation, andpyro-sequencing, use imaging of parallel cyclical chemical reactions.For example, reversible dye-terminators (RDTs) add onefluorescently-labeled nucleotide to a template (single-stranded DNA, forexample) per cycle and determine the type of incorporated nucleotidebased on the color of the fluorescent label. Such reactions requirechanging chemicals at every cycle and rely on fluidic cells to deliverthe chemicals to multiple reaction sites. Typically, each cycle of anRDT chemical reaction includes the steps of detritylation, coupling,capping and oxidation, and requires approximately 10-15 minutes.Additionally, the minimum distance between reaction sites is limited bythe optical resolution of fluorescent microscopes. Also, reaction sitescan be random or regular.

Accordingly, a need exists for decreasing the distance between reactionsites below the optical resolution limit so as to increase thethroughput of DNA sequencing.

SUMMARY

In one aspect of the present invention, a pipelined flow cell andrelated processes are provided. In one aspect of the invention, anexemplary apparatus can include an array comprising one or more pixels,wherein each of the one or more pixels comprises one or more reactionsites; and a network comprising (i) a set of one or more surfacechannels in a first component of the array, and (ii) a set of two ormore sub-surface channels in a second component of the array, whereineach reaction site corresponds to a channel from the set of one or moresurface channels, and wherein said set of two or more sub-surfacechannels comprises one or more sub-surface channels proximate to a firstportion of the array and one or more sub-surface channels proximate to asecond portion of the array. The apparatus also includes multiple viasin the second component of the array connecting each channel from theset of one or more surface channels to (i) one of the one or moresub-surface channels proximate to the first portion of the array and(ii) one of the one or more sub-surface channels proximate to the secondportion of the array.

An exemplary method can include steps of determining placement of one ormore reaction sites on a first component; providing a material for theone or more reaction sites in one or more surface channels of the firstcomponent; connecting the first component to a second component to forman array, wherein the one or more surface channels of the firstcomponent connect the one or more reaction sites with one or more vias,and wherein the second component comprises the one or more viasconnected to multiple sub-surface channels; and aligning the one or moresurface channels of the first component with the one or more vias of thesecond component to form a connection between the first component andthe second component.

In another aspect of the invention, an exemplary apparatus can includean array comprising one or more pixels, wherein each of the one or morepixels comprises multiple reaction sites openings; a first set of one ormore sub-surface channels, wherein each of the multiple reaction siteopenings is connected to a sub-surface channel from the first set of oneor more sub-surface channels; a second set of two or more sub-surfacechannels; and multiple vias connecting each channel from the first setof one or more sub-surface channels to (i) a first sub-surface channelfrom the second set of two or more sub-surface channels and (ii) asecond sub-surface channel from the second set of two or moresub-surface channels.

Yet another exemplary method can include steps of determining placementof multiple reaction site openings, wherein each reaction site openingis connected to a first sub-surface channel; connecting the firstsub-surface channel to two or more additional sub-surface channels bymultiple vias; and providing a material for multiple reaction sites,wherein an overlap of the multiple reaction site openings and thematerial delineate the multiple reaction sites.

Also, in yet another aspect of the invention, an exemplary method caninclude steps of delivering multiple items of chemical matterindependently to multiple reaction sites of a flow cell array acrossmultiple distinct instances of time; imaging multiple parallel chemicalreactions at the multiple reaction sites of the flow cell array; andrecording an emission from each of the multiple chemical reactions site.

Another aspect of the invention or elements thereof can be implementedin the to form of an article of manufacture tangibly embodying computerreadable instructions which, when implemented, cause a computer to carryout a plurality of method steps, as described herein. Furthermore,another aspect of the invention or elements thereof can be implementedin the form of an apparatus including a memory and at least oneprocessor that is coupled to the memory and configured to perform notedmethod steps. Yet further, another aspect of the invention or elementsthereof can be implemented in the form of means for carrying out themethod steps described herein, or elements thereof; the means caninclude hardware module(s) or a combination of hardware and softwaremodules, wherein the software modules are stored in a tangiblecomputer-readable storage medium (or multiple such media).

These and other objects, features and advantages of the presentinvention will become apparent from the following detailed descriptionof illustrative embodiments thereof, which is to be read in connectionwith the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating an existing sequencing setup;

FIG. 2 is a diagram illustrating cycles of a typical sequencingreaction;

FIG. 3 is a diagram illustrating time multiplexing between reactionsites, in accordance with an example embodiment of the invention;

FIG. 4 is a diagram illustrating time multiplexing between reactionsites with overlapping fluorescence at different sites, in accordancewith an example embodiment of the invention;

FIG. 5 is a diagram illustrating a top view of a pipelined microarraywith individually addressable reaction sites, in accordance with anexample embodiment of the invention;

FIG. 6a is a diagram illustrating exemplary placement of reaction sitesin a pixel using Gauss numbers, in accordance with an example embodimentof the invention;

FIG. 6b is a diagram illustrating exemplary placement of reaction sitesin a pixel using an approximate solution, in accordance with an exampleembodiment of the invention;

FIG. 7 is a diagram illustrating a side view of a two-componentmicroarray, in accordance with an example embodiment of the invention;

FIG. 8a is a diagram illustrating a cross-section view of an integratedmicroarray with two levels of fluidic channels, in accordance with anexample embodiment of the invention;

FIG. 8b is a diagram illustrating a side view of an integrated fluidiccell with two level fluidic channels, in accordance with an exampleembodiment of the invention;

FIG. 9 is a flow diagram illustrating techniques according to anembodiment of the invention;

FIG. 10 is a flow diagram illustrating techniques according to anembodiment of the invention;

FIG. 11 is a flow diagram illustrating techniques according to anembodiment of the invention; and

FIG. 12 is a system diagram of an exemplary computer system on which atleast one embodiment of the invention can be implemented.

DETAILED DESCRIPTION

As described herein, an aspect of the present invention includesproviding a pipelined flow cell microarray for imaging parallel chemicalreactions such as, for example, DNA sequencing. At least one embodimentof the invention includes generating and/or implementing a pipelinedpixel flow cell, wherein a number of sub-pixels are formed and chemistryis pipelined in each of the sub-pixels.

FIG. 1 is a diagram illustrating an existing sequencing setup. By way ofillustration, microarray 110, which includes multiple reaction sites120, is placed under an imaging microscope 130. Chemicals 140 are sentthrough the microarray 110 and leave the fluidic cell (as illustratedvia arrow 150).

FIG. 2 is a diagram illustrating cycles of a typical sequencing reaction210. During “Prepare” step 220, a set of chemicals is delivered to thefluidic cell and/or microarray (such as microarray 110 in FIG. 1) whichcauses some of the reaction sites (such as sites 120 in FIG. 1) tobecome fluorescent. During “Image” step 230, a fluorescent microscope(such as microscope 130 in FIG. 1) images the microarray. The computeranalysis of the emission from each reaction site provides informationabout the DNA sequence. For example, in the case of RDT, the color ofthe reaction site identifies the type of incorporated nucleotide. Insuch existing approaches, separating emissions from different test sitesis achieved through the use of sufficient distance between reactionsites, whereby the distance should exceed the resolution of the imagingsystem. Such a concept is referred to herein as space multiplexing.

Referring back to FIG. 2, “Prepare” step 220 and “Image” step 230comprise the first cycle 240 of the reaction 210. Cycles can be repeatedmany times, for example, to obtain the sequence of DNA molecules in eachreaction site.

Typically, “Prepare” step 220 is much longer in duration than “Image”step 230. Accordingly, this configuration can be used in accordance withat least one embodiment of the invention to implement time multiplexingbetween reaction sites. Assuming the reaction sites do not fluoresceduring “Prepare” steps, an example of such time multiplexing is depictedin FIG. 3.

Accordingly, FIG. 3 is a diagram illustrating time multiplexing betweenreaction sites, in accordance with an example embodiment of theinvention. Sequencing chemistry is independently delivered to eachreaction site, namely, Site₁ (350), Site₂, (360), Site₃ (370), andSite₄(380) with some time delay. As a result, each reaction site isfluorescent during non-overlapping time intervals, namely, Image₁-base₁(310), Image₂-base₁ (320), Image₃-base₁ (330), and Image₄-base₁ (340).Consequently, the emission from each reaction site can be recorded atdifferent time moments and there is no need to separate sites by asufficiently large distance. In other words, the distance betweenreaction sites can be made smaller than the optical resolution ofimaging system, provided that the chemistry is independently deliveredto these sites.

As further detailed herein, at least one embodiment of the invention canbegin executing a second instruction before a first instruction has beencompleted. As a result, several instructions can be in the pipeline atthe same time, each at a different execution step.

FIG. 4 is a diagram illustrating time multiplexing between reactionsites with overlapping fluorescence at different sites, in accordancewith an example embodiment of the invention. By way of illustration,FIG. 4 depicts an example wherein only two sites out of four arefluorescent at any given time moment. By way of example, at timeinterval t₁₁, only site₁ is fluorescent, and site₁ emits signal f₁₁. Themeasured signal from all four sites is s(t₁₁)=f₁₁. During time intervalt₁₂, site₂ becomes fluorescent, and site₂ emits signal f₂₁. The measuredsignal at this time interval is s(t₁₂)=f₁₁+f₂₁. During t₁₃, site₁ stopsfluorescence, but site₃, in turn, starts to emit signal f₃₁. As aresult, s(t₁₃)=f₃₁+f₃₁. Repeated cycling results in a system ofequations 410, which can be solved for individual emissions f_(ij) fromdifferent sites i=1 . . . 4 at different cycle numbers j=1, 2, . . . .It is noted that in accordance with at least one example embodiment ofthe invention, the maximum number of simultaneously fluorescent siteswill be limited by dynamic range of the imaging system.

Additionally, at least one embodiment of the invention includescombining space multiplexing and time multiplexing in a pipelinedmicroarray. As used herein, the term “pipelined” refers to aconfiguration for independently providing instructions to microfluidicchannels, as detailed herein.

FIG. 5 is a diagram illustrating a top view of a pipelined microarraywith individually addressable reaction sites, in accordance with anexample embodiment of the invention. In the example embodiment depictedin FIG. 5, the pipelined microarray is organized as an array of pixels510. In this specific example, a 2×2 array is depicted. It is to beappreciated, however, that this array is being described merely as anexample embodiment of the invention, and separate and/or additionalconfigurations can be implemented.

Pixel size can be determined, for example, by the optical resolution ofthe imaging microscope and can be of the order of a micrometer orlarger. Each pixel 510 has multiple reaction sites 520. In this specificexample depicted in FIG. 5, four reaction sites 520 are illustrated. Itis to again be appreciated, however, that this arrangement is describedmerely as an example, and separate and/or additional configurations canbe implemented in accordance with one or more embodiments of theinvention.

Additionally, microfluidic channels 530, aligned to the Y direction,connect to reaction sites 520. Such channels 530 are referred to hereinas Y-channels, and each reaction site has a corresponding Y-channel.Additionally, in at least one embodiment of the invention, a group ofmicrofluidic channels runs along the X direction (herein referred to asX-channels), wherein the group can include channels 540 below pixelarray and channels 550 above the pixel array. Further, in such anexample embodiment of the invention, it is assumed that intersecting X-and Y-channels do not form a fluidic connection. To form a connectionbetween intersecting X- and Y-channels, at least one embodiment of theinvention includes implementing and/or using fluidic vias 560.

The fluidic networks corresponding to different reaction sites 1 . . . 4(520) do not form fluidic connections. Accordingly, differentchemistries 570 can be simultaneously delivered to different reactionsites within a single pixel. Alternatively, the same chemistry can bedelivered to different reaction sites at different times, implementingtime multiplexing, such as illustrated in FIG. 3 and FIG. 4.

FIG. 6a is a diagram illustrating exemplary placement of reaction sitesin a pixel within a microarray 600 using Gauss numbers, in accordancewith an example embodiment of the invention. By way of example, in anillustrative embodiment of the invention, the placement of reactionsites must satisfy the following properties:

-   -   1) Each pixel 610 has equal number of reaction sites n;    -   2) Each reaction site 640 is connected to the plane as        illustrated in the figure to a microfluidic channel 620;    -   3) Microfluidic channels are parallel (either vertical or        horizontal) and do not overlap; and    -   4) The minimum distance 630 between any two reaction sites 640        is maximized.

These four conditions lead to an optimization problem on integerlattices. This problem has an exact optimal solution wherein n is aGaussian number, that is, when n represents a sum of two integersquares. By way of illustration, a list of such numbers for n≤30 is asfollows: {1, 2, 4, 5, 8, 9, 13, 16, 17, 20, 25, 26, 29}. FIG. 6aprovides an example of optimal reaction sites placement for n=5. Thegeneral solution for Gaussian case is as follows: Let n=a²+b². By way ofexample, the following situations are possible:

-   -   1) a≠0 and b≠0. As such, the optimal lattice of site placements        is given by the generator vectors (a/n, b/n) and (b/n, −a/n) on        the pixel grid, and has a minimum distance of 1/√n pixels.    -   2) n is a perfect square. As such, the optimal lattice of site        placements is given by the generator vectors (1, 0) and (1/√n,        1/n) on the pixel grid, and has a minimum distance of 1/√n        pixels.

If n is not a Gaussian number, an approximate solution with a minimumdistance sufficiently close to the optimal can be determined numericallyby exhaustive search through the n⁴ pairs of possible generating vectorswhich includes coordinates belonging to the set: {0, 1/n, 2/n, . . . ,(n−1)/n}. This computation can be carried out, for example, on acomputer for any n≤500. An example of an approximate solution for n=6 isillustrated in FIG. 6b . Accordingly, FIG. 6b is a diagram illustratingexemplary placement of reaction sites in a pixel within a microarray 601using an approximate solution, in accordance with an example embodimentof the invention. In such an example, the minimum distance between anytwo reaction sites is d≈0.3727 pixels, which is approximately 8.71%smaller than the Gaussian case ideal solution, for which the minimumdistance would be d=1/√6≈0.4082 pixels.

As detailed herein, a pipelined microarray, in accordance with one ormore embodiments of the invention, includes multiple unconnected fluidicnetworks. FIG. 7 is a diagram illustrating a side view of atwo-component microarray, in accordance with an example embodiment ofthe invention. By way of illustration, FIG. 7 depicts one exampleembodiment of such networks as a cross-section along line AB (as alsoillustrated in FIG. 5). Two independently-made components, firstcomponent 710 and second component 720, are bonded to form microarray730. Component 710 includes a surface microfluidic Y-channel 740, whichconnects reaction sites 750 with fluidic ports (vias) 760. As detailedherein, a reaction site (such as depicted by components 750 in FIG. 7)do not block the surface channel (such as channel 740), as each reactionsite serves as a chemical modification of the channel. Component 720includes sub-surface microfluidic X-channels 770. As illustrated in FIG.7, only X-channels aligned with fluidic vias (also referred to herein asports) 760 form a fluidic connection with Y-channel 740. Additionally,FIG. 7 depicts sub-surface channels 780 of the second component 720 thatconnect to surface channel 740 from the first component 710 throughfluidic via 760.

An integrated microarray flow cell can also be fabricated usingtechniques such as described, by way merely of example, in U.S.application Ser. No. 13/920,226, filed on Jun. 18, 2013 and entitled“Nanochannel Process and Structure for Bio-Detection,” which isincorporated by reference herein in its entirety.

FIG. 8a is a diagram illustrating a cross-section view of an integratedmicroarray with two levels of fluidic channels, in accordance with anexample embodiment of the invention. Such a structure as illustrated inFIG. 8a can be fabricated, for example, on a glass, silicon, or othersuitable substrate (such as further discussed below in connection withFIG. 8b ). With a silicon substrate or other material which would beetched by XeF₂, first a protection layer 810 of silicon dioxide would bedeposited. The first fluidic channel level 820 would be formed bydepositing an amorphous or polycrystalline silicon layer of the desiredchannel height, patterning the silicon (Si) layer into the desiredchannel configuration by a combination of photolithography and reactiveion etching (ME), stripping the photoresist and over-coating with a(thick) SiO₂ layer.

The fluidic via layer 830 could then be formed by planarizing the oxidelayer using chemical-mechanical polishing (CMP), either leaving thedesired thickness of oxide over the Si pattern that will become thefirst fluidic channel level, or depositing additional oxide after theplanarization step. Additionally, photolithography and ME can be used toremove the oxide layer over the first fluidic channel 820 where fluidicvias 830 are desired. The second fluidic channel level 840 is thenformed by depositing an amorphous or polycrystalline silicon layer ofthe desired channel height and patterning the Si layer into the desiredchannel configuration by a combination of photolithography and ME.Another oxide layer can also be deposited and planarized.

Further, an additional photolithography step can be used in combinationwith RIE etching of the oxide down to the silicon to create the fillports, reaction site openings 850, and vent holes. Fill ports are usedto introduce and remove the chemistry which is provided during use ofthe microarray flow cell, and vent holes are temporary openings usedduring XeF₂ etching. Vapor phase etching of the Si with XeF₂ can be usedto convert the Si structures into the integrated microarray flow cell.Also, an oxide deposition step can be performed to “pinch-off” and sealthe vent holes. The dimensions of the reaction site openings 850 andfill ports, as well as the thickness of the final oxide deposition step,are selected to not close off either the reaction site openings 850 orthe fill ports, while sealing the vent holes. For example, the reactionsites may be 0.5 microns on a side, the fill ports may be 2 millimeters(mm) in diameter, and the vent holes may be 0.1 microns on a side;accordingly, an oxide layer 0.1 microns thick would seal the vent holes,but not the other noted features. Further description of fill ports andvent holes can be found, by way of example, in U.S. application Ser. No.13/920,226, filed on Jun. 18, 2013 and entitled “Nanochannel Process andStructure for Bio-Detection,” which is incorporated by reference hereinin its entirety.

With the fabrication of an integrated microarray with two levels offluid channels described, a side view of such a microarray is shown inFIG. 8b . As further detailed herein, FIG. 8b depicts one exampleembodiment of such a microarray as a cross-section along line AB (asalso illustrated in FIG. 5). By way of illustration, FIG. 8b depicts anintegrated (single component) microarray 860 with two levels of fluidchannels: X-channels 870 and Y-channel 865. As depicted in FIG. 8b , theintegrated microarray 860 also includes fluid (or gaseous) vias 875positioned so as to form a connection between the X-channels 870 and theY-channel 865. Further, the integrated microarray 860 additionallyincludes reaction site openings 880, as well as a cover 885 (forexample, a glass cover) which may include a sealing polymer.

Accordingly, a number of options can be implemented to provide thenecessary or desired material at the reaction site openings 880. Forexample, one option is to deposit, on a cover glass 885, the desiredmaterials in the pattern shown in FIG. 6a and/or FIG. 6b , and to thenalign the active regions with the reaction site openings 880 down to theY-channel 865 in the integrated microarray 860. The cover glass 885 canbe temporally or permanently secured to the integrated microarray 860 toseal the reaction site openings 880.

If a temporary seal is used, the microarray can be reused. An exampleembodiment of the invention includes using a thin compliant polymerlayer on the cover glass 885 under the active materials so that a sealis formed when the cover glass 885 is compressed against the microarray860. The thickness of the cover glass can be, for example, on the orderof hundreds of micrometers (μm), and the thickness of a compliantpolymer will depend on the surface's flatness and can range from a fewto tens of microns. Note, as detailed above, that each reaction site canbe comprised of an array of reaction site openings so that an array ofreaction sites is formed even though the material provided on the coverglass is not patterned into a fine array. An alternate technique caninclude, for example, dissolving the necessary material into a wax orpolymer that could be dispensed as a liquid, which would wick into thereaction site openings and seal the openings.

FIG. 9 is a flow diagram illustrating techniques according to anembodiment of the invention. Step 902 includes determining placement ofone or more reaction sites on a first component. The determining stepcan be based on use of a Gauss number if the number of the one or morereaction sites represents a sum of two integer squares and/or based onuse of numerical approximation.

Step 904 includes providing a material for the one or more reactionsites in one or more surface channels of the first component. Step 906includes connecting the first component to a second component to form anarray, wherein the one or more surface channels of the first componentconnect the one or more reaction sites with one or more vias, andwherein the second component comprises the one or more vias connected tomultiple sub-surface channels. Step 908 includes aligning the one ormore surface channels of the first component with the one or more viasof the second component to form a connection between the first componentand the second component.

Also, the techniques depicted in FIG. 9 can include independentlydelivering chemical matter to each of the multiple reaction sites.

As also detailed herein, at least one embodiment of the inventionincludes an apparatus that includes an array comprising one or morepixels, wherein each of the one or more pixels comprises one or morereaction sites. Location of the one or more reaction sites on each ofthe one or more pixels can be based, for example, on utilization of oneor more Gauss numbers. The apparatus also includes a network comprising(i) a set of one or more surface channels in a first component of thearray, and (ii) a set of two or more sub-surface channels in a secondcomponent of the array, wherein each reaction site corresponds to achannel from the set of one or more surface channels, and wherein saidset of two or more sub-surface channels comprises one or moresub-surface channels proximate to a first portion of the array and oneor more sub-surface channels proximate to a second portion of the array.The apparatus further includes multiple vias in the second component ofthe array connecting each channel from the set of one or more surfacechannels to (i) one of the one or more sub-surface channels proximate tothe first portion of the array and (ii) one of the one or moresub-surface channels proximate to the second portion of the array.

As detailed herein, the network can specifically include a set of one ormore fluidic channels and a set of two or more fluidic channels, andwherein said multiple vias comprise multiple fluidic vias. Similarly, atleast one embodiment of the invention can also include a network thatincludes a set of one or more gaseous channels and a set of two or moregaseous channels, and wherein said multiple vias comprise multiplegaseous vias.

Additionally, in such an apparatus, the size of each of the multiplepixels is based on an optical resolution of an imaging device associatedwith the apparatus. By way of example, the size of each of the multiplepixels can be approximately one micrometer, or can be greater than onemicrometer. Further, in at least one embodiment of the invention, eachof the multiple pixels includes an equal number of reaction sites.

Also, in at least one embodiment of the invention, the one or moresurface channels are parallel and do not overlap, the two or moresub-surface channels are parallel and do not overlap, and the set of oneor more surface channels runs perpendicular to the set of two or moresub-surface channels.

FIG. 10 is a flow diagram illustrating techniques according to anembodiment of the present invention. Step 1002 includes determiningplacement of multiple reaction site openings, wherein each reaction siteopening is connected to a first sub-surface channel. The determiningstep can be based on use of a Gauss number if the number of the multiplereaction site openings represents a sum of two integer squares and/orbased on use of numerical approximation.

Step 1004 includes connecting the first sub-surface channel to two ormore additional sub-surface channels by multiple (fluidic) vias. Step1006 includes providing a material for multiple reaction sites, whereinan overlap of the multiple reaction site openings and the materialdelineate the multiple reaction sites.

The techniques depicted in FIG. 10 can also include incorporating acover to seal the multiple reaction site openings. The cover caninclude, for example, a glass cover with or without a sealing polymer.Additionally, incorporating the cover can include temporarily securingthe cover or permanently securing the cover. Also, the techniquesdepicted in FIG. 10 can include independently delivering chemical matterto each of the multiple reaction sites.

Additionally, at least one embodiment of the invention includes anapparatus that includes an array comprising one or more pixels, whereineach of the one or more pixels comprises multiple reaction sitesopenings, and a first set of one or more sub-surface channels, whereineach of the multiple reaction site openings is connected to asub-surface channel from the first set of one or more sub-surfacechannels. The apparatus additionally includes a second set of two ormore sub-surface channels, and multiple vias connecting each channelfrom the first set of one or more sub-surface channels to (i) a firstsub-surface channel from the second set of two or more sub-surfacechannels and (ii) a second sub-surface channel from the second set oftwo or more sub-surface channels. Such an apparatus can additionallyinclude a cover secured (temporarily or permanently) to the apparatus.The cover can include, for example, a glass cover with or without asealing polymer.

In such an apparatus, each of the pixels is configured to independentlyreceive chemical matter at each of the multiple reaction site openings.Additionally, the size of each pixel can be based on an opticalresolution of an imaging device associated with the apparatus. Further,in such an apparatus, each of the multiple reaction sites openings caninclude an array of multiple openings, and the location of each of themultiple reaction sites openings on each of the one or more pixels canbe based on utilization of one or more Gauss numbers.

FIG. 11 is a flow diagram illustrating techniques according to anembodiment of the invention. Step 1102 includes delivering multipleitems of chemical matter independently to multiple reaction sites of aflow cell array across multiple distinct instances of time. The multipledistinct instances of time can include multiple non-overlappinginstances of time determined based on the multiple parallel chemicalreactions and/or based on the multiple items of chemical matter.

Step 1104 includes imaging multiple parallel chemical reactions at themultiple reaction sites of the flow cell array. The multiple parallelchemical reactions can include, for example, multiple instances ofbiological sequencing (such as DNA sequencing). Step 1106 includesrecording an emission from each of the multiple chemical reactions site.

The techniques depicted in one or more of the above-detailed flowdiagrams can also, as described herein, include providing a system,wherein the system includes distinct software modules, each of thedistinct software modules being embodied on a tangible computer-readablerecordable storage medium. All of the modules (or any subset thereof)can be on the same medium, or each can be on a different medium, forexample. The modules can include any or all of the components shown inthe figures and/or described herein. In an aspect of the invention, themodules can run, for example, on a hardware processor. The method stepscan then be carried out using the distinct software modules of thesystem, as described above, executing on a hardware processor. Further,a computer program product can include a tangible computer-readablerecordable storage medium with code adapted to be executed to carry outat least one method step described herein, including the provision ofthe system with the distinct software modules.

Additionally, the techniques depicted in one or more of theabove-detailed flow diagrams can be implemented via a computer programproduct that can include computer useable program code that is stored ina computer readable storage medium in a data processing system, andwherein the computer useable program code was downloaded over a networkfrom a remote data processing system. Also, in an aspect of theinvention, the computer program product can include computer useableprogram code that is stored in a computer readable storage medium in aserver data processing system, and wherein the computer useable programcode is downloaded over a network to a remote data processing system foruse in a computer readable storage medium with the remote system.

An aspect of the invention or elements thereof can be implemented in theform of an apparatus including a memory and at least one processor thatis coupled to the memory and configured to perform exemplary methodsteps.

Additionally, an aspect of the present invention can make use ofsoftware running on a general purpose computer or workstation. Withreference to FIG. 12, such an implementation might employ, for example,a processor 1202, a memory 1204, and an input/output interface formed,for example, by a display 1206 and a keyboard 1208. The term “processor”as used herein is intended to include any processing device, such as,for example, one that includes a CPU (central processing unit) and/orother forms of processing circuitry. Further, the term “processor” mayrefer to more than one individual processor. The term “memory” isintended to include memory associated with a processor or CPU, such as,for example, RAM (random access memory), ROM (read only memory), a fixedmemory device (for example, hard drive), a removable memory device (forexample, diskette), a flash memory and the like. In addition, the phrase“input/output interface” as used herein, is intended to include, forexample, a mechanism for inputting data to the processing unit (forexample, mouse), and a mechanism for providing results associated withthe processing unit (for example, printer). The processor 1202, memory1204, and input/output interface such as display 1206 and keyboard 1208can be interconnected, for example, via bus 1210 as part of a dataprocessing unit 1212. Suitable interconnections, for example via bus1210, can also be provided to a network interface 1214, such as anetwork card, which can be provided to interface with a computernetwork, and to a media interface 1216, such as a diskette or CD-ROMdrive, which can be provided to interface with media 1218.

Accordingly, computer software including instructions or code forperforming the methodologies of the invention, as described herein, maybe stored in associated memory devices (for example, ROM, fixed orremovable memory) and, when ready to be utilized, loaded in part or inwhole (for example, into RAM) and implemented by a CPU. Such softwarecould include, but is not limited to, firmware, resident software,microcode, and the like.

A data processing system suitable for storing and/or executing programcode will include at least one processor 1202 coupled directly orindirectly to memory elements 1204 through a system bus 1210. The memoryelements can include local memory employed during actual implementationof the program code, bulk storage, and cache memories which providetemporary storage of at least some program code in order to reduce thenumber of times code must be retrieved from bulk storage duringimplementation.

Input/output or I/O devices (including but not limited to keyboards1208, displays 1206, pointing devices, and the like) can be coupled tothe system either directly (such as via bus 1210) or through interveningI/O controllers (omitted for clarity).

Network adapters such as network interface 1214 may also be coupled tothe system to enable the data processing system to become coupled toother data processing systems or remote printers or storage devicesthrough intervening private or public networks. Modems, cable modems andEthernet cards are just a few of the currently available types ofnetwork adapters.

As used herein, including the claims, a “server” includes a physicaldata processing system (for example, system 1212 as shown in FIG. 12)running a server program. It will be understood that such a physicalserver may or may not include a di splay and keyboard.

As will be appreciated by one skilled in the art, aspects of the presentinvention may be embodied as a system, method and/or computer programproduct. Accordingly, aspects of the present invention may take the formof an entirely hardware embodiment, an entirely software embodiment(including firmware, resident software, micro-code, etc.) or anembodiment combining software and hardware aspects that may allgenerally be referred to herein as a “circuit,” “module” or “system.”Furthermore, as noted herein, aspects of the present invention may takethe form of a computer program product that may include a computerreadable storage medium (or media) having computer readable programinstructions thereon for causing a processor to carry out aspects of thepresent invention.

The computer readable storage medium can be a tangible device that canretain and store instructions for use by an instruction executiondevice. The computer readable storage medium may be, for example, but isnot limited to, an electronic storage device, a magnetic storage device,an optical storage device, an electromagnetic storage device, asemiconductor storage device, or any suitable combination of theforegoing. A non-exhaustive list of more specific examples of thecomputer readable storage medium includes the following: a portablecomputer diskette, a hard disk, a random access memory (RAM), aread-only memory (ROM), an erasable programmable read-only memory (EPROMor Flash memory), a static random access memory (SRAM), a portablecompact disc read-only memory (CD-ROM), a digital versatile disk (DVD),a memory stick, a floppy disk, a mechanically encoded device such aspunch-cards or raised structures in a groove having instructionsrecorded thereon, and any suitable combination of the foregoing. Acomputer readable storage medium, as used herein, is not to be construedas being transitory signals per se, such as radio waves or other freelypropagating electromagnetic waves, electromagnetic waves propagatingthrough a waveguide or other transmission media (for example, lightpulses passing through a fiber-optic cable), or electrical signalstransmitted through a wire.

Computer readable program instructions described herein can bedownloaded to respective computing/processing devices from a computerreadable storage medium or to an external computer or external storagedevice via a network, for example, the Internet, a local area network, awide area network and/or a wireless network. The network may comprisecopper transmission cables, optical transmission fibers, wirelesstransmission, routers, firewalls, switches, gateway computers and/oredge servers. A network adapter card or network interface in eachcomputing/processing device receives computer readable programinstructions from the network and forwards the computer readable programinstructions for storage in a computer readable storage medium withinthe respective computing/processing device.

Computer readable program instructions for carrying out operations ofthe present invention may be assembler instructions,instruction-set-architecture (ISA) instructions, machine instructions,machine dependent instructions, microcode, firmware instructions,state-setting data, or either source code or object code written in anycombination of one or more programming languages, including an objectoriented programming language such as Java, Smalltalk, C++ or the like,and conventional procedural programming languages, such as the “C”programming language or similar programming languages. The computerreadable program instructions may execute entirely on the user'scomputer, partly on the user's computer, as a stand-alone softwarepackage, partly on the user's computer and partly on a remote computeror entirely on the remote computer or server. In the latter scenario,the remote computer may be connected to the user's computer through anytype of network, including a local area network (LAN) or a wide areanetwork (WAN), or the connection may be made to an external computer(for example, through the Internet using an Internet Service Provider).In some embodiments, electronic circuitry including, for example,programmable logic circuitry, field-programmable gate arrays (FPGA), orprogrammable logic arrays (PLA) may execute the computer readableprogram instructions by utilizing state information of the computerreadable program instructions to personalize the electronic circuitry,in order to perform aspects of the present invention.

Aspects of the present invention are described herein with reference toflowchart illustrations and/or block diagrams of methods, apparatus(systems), and computer program products according to embodiments of theinvention. It will be understood that each block of the flowchartillustrations and/or block diagrams, and combinations of blocks in theflowchart illustrations and/or block diagrams, can be implemented bycomputer readable program instructions.

These computer readable program instructions may be provided to aprocessor of a general purpose computer, special purpose computer, orother programmable data processing apparatus to produce a machine, suchthat the instructions, which execute via the processor of the computeror other programmable data processing apparatus, create means forimplementing the functions/acts specified in the flowchart and/or blockdiagram block or blocks. These computer readable program instructionsmay also be stored in a computer readable storage medium that can directa computer, a programmable data processing apparatus, and/or otherdevices to function in a particular manner, such that the computerreadable storage medium having instructions stored therein comprises anarticle of manufacture including instructions which implement aspects ofthe function/act specified in the flowchart and/or block diagram blockor blocks.

The computer readable program instructions may also be loaded onto acomputer, other programmable data processing apparatus, or other deviceto cause a series of operational steps to be performed on the computer,other programmable apparatus or other device to produce a computerimplemented process, such that the instructions which execute on thecomputer, other programmable apparatus, or other device implement thefunctions/acts specified in the flowchart and/or block diagram block orblocks.

The flowchart and block diagrams in the Figures illustrate thearchitecture, functionality, and operation of possible implementationsof systems, methods, and computer program products according to variousembodiments of the present invention. In this regard, each block in theflowchart or block diagrams may represent a module, segment, or portionof instructions, which comprises one or more executable instructions forimplementing the specified logical function(s). In some alternativeimplementations, the functions noted in the block may occur out of theorder noted in the figures. For example, two blocks shown in successionmay, in fact, be executed substantially concurrently, or the blocks maysometimes be executed in the reverse order, depending upon thefunctionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts or carry out combinations of special purpose hardwareand computer instructions.

It should be noted that any of the methods described herein can includean additional step of providing a system comprising distinct softwaremodules embodied on a computer readable storage medium; the modules caninclude, for example, any or all of the components detailed herein. Themethod steps can then be carried out using the distinct software modulesand/or sub-modules of the system, as described above, executing on ahardware processor 1202. Further, a computer program product can includea computer-readable storage medium with code adapted to be implementedto carry out at least one method step described herein, including theprovision of the system with the distinct software modules.

In any case, it should be understood that the components illustratedherein may be implemented in various forms of hardware, software, orcombinations thereof, for example, application specific integratedcircuit(s) (ASICS), functional circuitry, an appropriately programmedgeneral purpose digital computer with associated memory, and the like.Given the teachings of the invention provided herein, one of ordinaryskill in the related art will be able to contemplate otherimplementations of the components of the invention.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition ofanother feature, integer, step, operation, element, component, and/orgroup thereof.

The corresponding structures, materials, acts, and equivalents of allmeans or step plus function elements in the claims below are intended toinclude any structure, material, or act for performing the function incombination with other claimed elements as specifically claimed.

At least one aspect of the present invention may provide a beneficialeffect such as, for example, increasing the density of chemicalreactions.

The descriptions of the various embodiments of the present inventionhave been presented for purposes of illustration, but are not intendedto be exhaustive or limited to the embodiments disclosed. Manymodifications and variations will be apparent to those of ordinary skillin the art without departing from the scope and spirit of the describedembodiments. The terminology used herein was chosen to best explain theprinciples of the embodiments, the practical application or technicalimprovement over technologies found in the marketplace, or to enableothers of ordinary skill in the art to understand the embodimentsdisclosed herein.

What is claimed is:
 1. A method comprising: positioning multiplereaction sites in a first set of surface channels within a physicalelement flow cell array, wherein the first set of surface channels arealigned to a first direction, and wherein positioning comprisespositioning the multiple physical reaction sites within the first set ofsurface channels such that a distance between each of the multiplephysical reaction sites is smaller than an optical resolutionperpendicular to the optical path of a given fluorescence microscope,and wherein the distance is less than one micron; connecting the firstset of surface channels to a second set of surface channels by multiplevias, wherein the second set of surface channels are aligned to a seconddirection; establishing a designation wherein pairs of surface channelswithin the second set are designated for use with (i) a distinct one ofmultiple chemical matters, (ii) one or more reaction sites, among themultiple reaction sites, designated for use with the distinct one of themultiple chemical matters, and (iii) one or more surface channels withinthe first set connected to the one or more reaction sites designated foruse with the distinct one of the multiple chemical matters; delivering,within at least a portion of the physical element flow cell array, themultiple chemical matters to the multiple reaction sites; imaging,subsequent to said delivering, multiple parallel chemical reactions atthe multiple reaction sites using the given fluorescence microscope,wherein the multiple parallel chemical reactions comprise multipleinstances of biological sequencing, and wherein the distance between twoor more of the multiple reaction sites being imaged simultaneously isgreater than the optical resolution of the given fluorescence microscopewhile the distance between two or more of the other multiple physicalreaction sites is less than the optical resolution of the givenfluorescence microscope and less than one micron; and incorporating acover in connection with the multiple reaction sites.
 2. The method ofclaim 1, wherein said incorporating comprises securing the cover.